Specificity of anti-HLA-B27 cytotoxic T lymphocytes

Sub-types of HLA-B27 were detected by cytotoxic T lymphocytes (CTL) generated between HLA-A, -B- and -C-identical B27-positive individuals. We now report the specificity of six independent CTL's generated by mixed lymphocyte culture (MLC) of HLA-A, -B and -C serologically identical B27-positive responder and stimulator cells. Three CTL's recognize one sub-type, and three the other. The combined reactivity of all CTL's allows unequivocal "typing" of B27-positive cells for the two different sub-types B27K and B27W. The specificity of two CTL's was analysed by cold-target inhibition. The results indicate that (1) no further sub-types of HLA-B27 can be detected by the CTL's raised in these combinations; (2) the majority of the CTL's is directed against the B27 antigens; and (3) "extra reactions" on B27-negative cells are caused by a subset(s) of CTL's recognizing unknown antigens shared between stimulator and target cells. CTL's raised by stimulation of HLA-B27-negative responder cells with B27-positive cells of either sub-type lysed all B27-positive target cells indiscriminately. In cold-target inhibition, however, B27-positive cells, carrying the sub-type of B27 different from that of the stimulator, could not inhibit the lysis of cells bearing the stimulator sub-type of B27. This indicates the activation, in B27-negative responders, of at least two different groups of CTL clones, one directed against shared determinants of HLA-B27, and one against the HLA-B27 sub-type. Heterogeneity of the HLA-B27 antigen may have implications for studies on the well-known association between this antigen and various diseases.     

Possible mechanisms by which alloantisera inhibit in the MLC test

MLC inhibition studies were performed with two human alloantisera: one specific for HLA-B7, the other for HLA-DRw7. The stimulator cell inhibitory effects of these sera were tested in primary and secondary MLC tests. Both sera inhibited in the primary MLC, whereas only the anti-DRw7 serum was capable of blocking the secondary MLC test. The difference in inhibiting properties of these sera was further analyzed in primary MLC tests using selected MLC combinations, Fc receptor negative cell populations and pepsin digests of the anti-B7 serum. Anti-DRw7 antibodies could inhibit by masking the stimulatory DR antigens. The inhibition of the anit-B7 antiserum was dependent on Fc, which suggested that anti HLA-B antibodies inhibited by some other mechanism. This inhibition could have been caused by antibody dependent cellular lympholysis of the stimulator cells or by the induction of suppressor cell activity.     

Unbalanced regulation of the expression of HLA-A and -B antigens in metastatic melanoma cells compared to autologous PBMC: A quantitative analysis of fourteen patients.

The quantitative cell surface expression of the gene products of HLA-A and -B loci is genetically predetermined (following Mendelian inheritance) in a given individual; in addition, the regulation of their expression is tightly coordinated since the ratio of HLA-A and -B antigens expression is constant on different cell types and the expression of HLA-B antigens is lower than that of HLA-A antigens. In view of these considerations and of the potential relevance of the quantitative expression of the gene products of HLA-A and -B loci in antigen presentation and for T cell-based specific immunotherapy, levels of cell surface HLA-A (mAb A131) and -B (mAb YTH) antigens were investigated by flow cytometry on fourteen primary cultures of melanoma cells (analyzed between in vitro passages 5 to 10) derived from unrelated melanoma patients and compared to those obtained with autologous PBMC. All melanoma cells and PBMC investigated were stained by mAb A131 and YTH (samples were considered positive when the mean value of fluorescence intensity with specific mAb was at least double than negative control mAb). The mean values of mean fluorescence intensity obtained for HLA-A and HLA-B antigens were 275±247 and 35±30 on melanoma cells and 1520±490 and 780±340 on PBMC and were both significantly different in a paired test between melanoma cells and PBMC (HLA-A, P=2×10⁻⁶; HLA-B, P=1×10⁻⁶); thus, the expression of HLA-A and -B antigens was 5.5 and 22.1 times lower on melanoma cells than on autologous PBMC. Simple linear regression analysis showed a high correlation (r=0.9; P=1×10⁻⁵) between the mean values of fluorescence intensity observed for HLA-A and -B antigens in PBMC; in contrast, a low correlation (r=0.6; P=1×10⁻²) was found in melanoma cells. Therefore, we calculated the ratio between the mean values of mean fluorescence intensity obtained for HLA-A and -B antigens in melanoma cells and autologous PBMC. The ratio HLA-A vs HLA-B was 10.9±8.0 (range: 2.1 to 32.6) and 2.1±0.7 (range: 1.28 to 3.58) in melanoma cells and PBMC, respectively (p=1×10⁻³, paired t test). Results similar to those obtained with mAb YTH were also obtained with the anti-HLA-B antigens mAb Q6/64 and H2-89-1. Our data, altogether, strongly suggest the existance of an alteration involving the coordinated regulation of the expression of HLA-A and HLA-B loci in melanoma cells.     

Characteristics of graft-infiltrating lymphocytes after human heart transplantation HLA mismatches and the cellular immune response within the transplanted heart

The influence of HLA mismatches between donor and recipient on the phenotypes, function, and specificity of T-lymphocyte cultures derived from endomyocardial biopsies was studied in 118 heart transplant recipients. In case of HLA-DR mismatches, the majority of the EMB-derived cultures were dominated by CD4⁺ T cells while, in patients with HLA-A and -B mismatches but without DR mismatches, CD8⁺ T cells comprised the predominant T-cell subset. Cytotoxicity against donor antigens was observed in 75% of the cultures. A significantly (p < 0.005) lower proportion of the cultures showed cytotoxicity against HLA-A antigens (36%) when compared with HLA-B (53%) or HLA-DR (49%). An HLA-A2 mismatch elicited a cytotoxic response that was comparable to that found against HLA-B and -DR antigens: 62% of the cultures from HLA-A2 mismatched donor-recipient combinations was reactive against A2. A higher number of A, B, or DR mismatches resulted in a higher number of cytotoxic cultures directed against these antigens. A higher number of HLA-B and -DR mismatches was associated with a lower freedom from rejection. Our data indicate that, despite the use of adequate immunosuppressive therapy, the degree of HLA matching plays a crucial role in the immune response against a transplanted heart, resulting in a significant effect on freedom from rejection.     

Inhibition of responder cell activity in mixed leukocyte culture reactions

An HLA-B7 antiserum showed cross-reactivity with HLA-B8, Bw41, a split of B40 (Bw60) and possibly Bw22 and B27, thus detecting one or more determinants on these antigens similar to an antigenic site on HLA-B7 usually not detected by other B7-antisera. The cross-reactions were demonstrable by adsorption of antibodies on lymphocytes followed by release at 37 degrees C, which enabled the detection of weak and otherwise hardly detectable reactivity. Released antibody molecules were detected in two different assays: (1) Antibody-dependent cellular cytotoxicity (ADCC) with HLA-B7 positive target cells (fluorochromasia micro ADCC). (2) Inhibition of MLC reactions with B7 positive stimulator cells. The B7-antibody, as detected in both assays, was released in decreasing activity from Bw41 greater than B8 greater than Bw60 much greater than B7 greater than (B27 = Bw22) positive cells. The order of sensitivity in which the various antigens were detected in ADCC assays in which the antiserum activity was measured directly on various target cells was different, viz. HLA-B7 greater than Bw60 = B27 greater than Bw41 greater than B8. Bw22 was not detected. Absorption studies demonstrated that HLA-B7 positive cells bound more B7 antibody activity than B8 positive cells. However, antibody molecules bound to B7 positive cells were mainly released as immune complexes, which could be dissociated by treatment with acid. In contrast, B7 antibody molecules bound to B8 positive cells were released as free antibody molecules. This marked difference in shedding properties further explained the previously described B7 specific unresponsiveness in MLC of HLA-B8 (and also Bw41) positive responder cells after sensitization with the B7 antiserum (de Rooij et al. 1980).     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.     

ECDI偶联的人抗原提呈细胞诱导供者抗原特异性CD8+ T细胞耐受的实验研究

目的：研究体外模拟异基因移植环境下,碳二亚胺（ECDI）偶联的供者抗原提呈细胞（ECDI-APCs）诱导受者T细胞针对供者抗原特异性耐受的效果。方法：每组以HLA-A、-B、-DR完全错配的3名志愿者外周血淋巴细胞建立混合淋巴细胞培养体系,模拟异基因移植环境。在-6 d将受者外周血单个核细胞（PBMCs）与供者ECDI-APCs共培养,第0天再次加入新鲜分离的原供者APCs或无关供者APCs模拟移植,第8天以流式细胞术检测受者T细胞的增殖情况。结果：ECDI偶联浓度为150 mg/ml,供、受者细胞共培养比例为0.1∶1时,ECDI-APCs可诱导出受者T细胞对原供者抗原刺激的耐受状态（P<0.05）,其中受者CD8⁺ T细胞的耐受具有供者抗原特异性（P<0.05）,而CD4⁺ T细胞的耐受无抗原特异性（P>0.05）。结论：ECDI-APCs能诱导CD8⁺ T细胞对同种异体抗原刺激的耐受状态,并且具有供者抗原特异性,可为临床器官移植术后供者抗原特异性耐受的建立提供实验依据。     

Human mumps virus-specific cytotoxic T lymphocytes: quantitative analysis of HLA restriction

To obtain quantitative information about the use of HLA antigens as restriction element by antiviral cytotoxic T lymphocytes (CTL), we have analyzed precursors of human mumps virus-specific CTL by limiting dilution. CTL generated by restimulation of peripheral blood T lymphocytes with autologous mumps virus (MV)-infected stimulator cells were restricted by autologous HLA class I antigens, and derived from the T4-8+ population. They were specific for MV and did not lyse autologous target cells infected with other viruses. Frequencies of MV-specific CTL precursors ranged from 1/500 to 1/8000. HLA restriction was analyzed by split-well analysis of individual CTL colonies. CTL recognizing HLA-A or B antigens were unequally distributed: HLA-B7, -B13, and -B27 were found to function as predominant, in some cases as exclusive, restriction elements, whereas other antigens such as HLA-A24 were never or rarely used. In several combinations, there was no evidence for antigenic variants of HLA molecules as reason for the failure to be recognized. The proportion of CTL precursors recognizing HLA-A2 and -B8 seemed to be dependent on the presence or absence of "dominant" restriction elements. We conclude that CTL precursors recognizing certain virus-HLA combinations are preferentially expanded during an infection, but that low responsiveness to a given combination is not necessarily absolute.     

Antibodies against donor antigens on endothelial cells and monocytes in eluates of rejected kidney allografts

Acidic eluates from two rejected kidney allografts contained antibodies reacting only with the endothelial cells and the monocytes of the specific kidney donors. No reaction was observed with the B-cells or the T-cells of these donors. When these eluates were tested against the leukocytes of healthy blood donors that were typed for HLA-A, -B, -C and DR, positive reactions were observed with the monocyte but not with the lymphocyte fraction. The first eluate reacted with 6.4% of the donors and the second eluate with 38.3%. No correlation with any of the HLA-A, -B, -C or DR antigens was found. If a more extended study proves that the endothelial-monocyte antigens do play an important role in kidney transplantation, matching of donors and recipients for these antigens and cross-matching recipient serum with donor monocytes may become a necessity in the future.     

Role of blood transfusions on the induction of antibodies against recognition sites on T lymphocytes in renal transplant patients

We have tested sera from 23 renal allograft recipients to study the effects of blood transfusions on the induction of antibodies directed against recognition sites on T lymphocytes. The results demonstrate that antibodies capable of inhibiting responses in MLC could be induced by blood transfusion. This inhibition in MLC is observed by treatment of responder lymphocytes with serum plus rabbit complement and is mediated by IgG antibodies. Also, the inhibitory effect is specific for certain responder cells and is not mediated by antibodies against common surface antigens of either the responder or the stimulator lymphocytes. The antibodies inhibiting proliferative responses in MLC against antigens present on the kidney donor were demonstrable in renal transplant recipients with functional allografts, but not in patients who had rejected the graft. The data suggest that antibodies directed against recognition sites on T lymphocytes could be induced by blood transfusions and these antibodies may be associated with prolonged graft survival.     

T lymphocyte subsets in human intestinal mucosa: the distribution and relationship to MHC-derived antigens

T lymphocytes in the normal human intestinal tract have been analysed in tissue sections by a double-marker immunofluorescence technique, combining antiserum to T lymphocyte antigen (HuTLA) with a monoclonal antibody detecting T cells of suppressor-cytotoxic phenotype (OKT8). The distribution of HLA-A -B, -C and Ia-like antigens in intestinal mucosa was also examined by a similar method. In small and large intestine 67 to 90% (mean 70%) of intraepithelial T lymphocytes were of suppressor-cytotoxic phenotype (OKT8+). In contrast, only 27 to 56% (mean 39%) of lamina propria T cells were OKT8+. Intestinal epithelial cells demonstrated strong membrane staining for HLA-A, -B, -C antigens. Ia-like antigens were detected on the epithelial cells of small intestinal villi, but not on colonic epithelial cells. Lamina propria macrophages expressed both HLA-A, -B, -C and Ia-like antigens, the latter having strong membrane and cytoplasmic fluorescence. The distribution of T cells with suppressor-cytotoxic or inducer phenotype in the intestinal epithelium and lamina propria may be related to the differential expression of Ia-like and HLA-A, -B, -C antigens in intestinal mucosa.     

An antigen processing polymorphism revealed by HLA-B8-restricted cytotoxic T lymphocytes which does not correlate with TAP gene polymorphism

In previous studies of antigen presentation through HLA-B27, we identified a healthy person whose lymphoblastoid cells do not present three B27-restricted viral epitopes to specific cytotoxic T lymphocytes (CTL), despite adequate cell surface expression of HLA-B2702 of normal sequence. Similar findings were observed in all members of his family sharing the HLA-A3-B2702 haplotype. The original donor, NW, carries HLA-B8 on his other class I haplotype, which his daughter, HW, has inherited. We now report a failure to present an HLA-B8-restricted epitope from influenza nucleoprotein following viral infection of NW cells, although exogenous added peptide is still presented normally. However, cells from HW, which do not carry the A3-B2702 haplotype, present the expected epitope after viral infection. Another B8-restricted epitope, from human immunodeficiency virus-gag, is presented equally well by both cell lines when infected with gag-vaccinia. This antigen processing phenotype does not correlate with any of the known human TAP-1 and TAP-2 polymorphisms.     

Comparison of serologic and biochemical definitions of HLA class I polymorphisms in cadaver renal transplantation

The role of biochemical variants in matching renal donors with recipients was investigated in a population of 26 donor recipient pairs from the period 1979-85. Serologic typing was compared with a biochemical analysis of HLA-A and -B antigens in a group in which there had been no mismatches for HLA-A and -B antigens by serology. HLA-A and -B serology was updated using stored material for 92.2% of the renal recipients and donors (n = 51), and HLA-A, -B, and -DR serology was updated for 84.3%. No inconsistencies were found with the HLA-A and -B antigens assigned at the time of transplant. HLA-A and -B antigens of 70% of the renal recipients and donors were analyzed by immunoprecipitation from lymphocytes and serum and analyzed after isoelectric focusing by autoradiography and Western blotting. No biochemical differences between recipient and donor were found for any one pair when HLA-A and -B antigens were serologically the same. This biochemical analysis was useful in providing results where there was doubt over the presence or absence of an antigen serologically. In a well-matched situation, HLA-A and -B serologic types were concordant with results obtained biochemically. However, in a poorly matched situation this could be different.     

Expression of HLA-A and -B antigens on differentiating U-937 cells

Cells from the human immature monocytoid cell line U-937 were induced with 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) to differentiate towards macrophage-like cells. The expression of HLA-antigens during differentiation was examined with a panel of monoclonal antibodies directed against monomorphic and polymorphic determinants. Class II antigens could be detected neither on uninduced nor on TPA-induced U-937 cells. While the expression of HLA-A3 did not change significantly during differentiation, the "supertypic" specificities HLA-Bw4 and Bw6 as well as the "private" specificity HLA-B18 could be detected only on a drastically decreased number of cells after 4 days of exposure to TPA. This may imply a selective loss of HLA-B molecules from the cell membrane and therefore a separate regulatory control of HLA-A and -B antigens.     

Failure of Epstein-Barr virus-specific cytotoxic T lymphocytes to lyse B cells transformed with the B95-8 strain is mapped to an epitope that associates with the HLA-B8 antigen.

There are two types, A and B, of Epstein-Barr virus (EBV) and B95-8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV-specific cytotoxic T lymphocytes (CTL) generated in short-term cultures following stimulation with the autologous B95-8-transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95-8 isolate did not lyse haploidentical B95-8 LCL expressing the HLA-A1, -B8, -DR3 paternal haplotype. In contrast, the haploidentical B95-8 LCL expressing the HLA-A11, -B51, -DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95-8 LCL expressing the haploidentical HLA-A1, -B8, -DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV-specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA-A11, -B51, -DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV-specific response. CTL clones were generated from two HLA-B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross-reactive for an immunodominant B95-8-associated peptide epitope that interacted with the HLA-B8 allele but failed to lyse B95-8-transformed LCL targets unless the targets were pre-coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA-B8+ donor, but failed to lyse the B95-8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95-8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögren''s syndrome which is closely associated with HLA-B8.     

Detection of HLA antigens on blood platelets and lymphocytes by means of monoclonal antibodies in an ELISA technique

The expression of HLA-A and -B antigens on peripheral blood lymphocytes and blood platelets was measured using monoclonal antibodies in a semi-quantitative ELISA technique. Reactively of monoclonal anti-HLA-A2 and anti-HLA-B7, with lymphocytes as well as platelets, was in agreement with the presence of these antigens as detected by conventional HLA typing of lymphocytes. When the actual amount of HLA antigens was measured, a gene-dose effect was seen: cells from HLA-B7-homozygous individuals bound more monoclonal anti-HLA-B7 antibodies compared to their HLA-B7-heterozygous siblings. At the same time, cells of different donors showed only very small differences in binding of monoclonal antibody against an HLA-"backbone" determinant. Relative to total HLA-A, -B and -C expression, the amounts of HLA-A2 on lymphocytes and platelets were similar. On the other hand, the expression of HLA-B7 on platelets was diminished compared to that on peripheral blood lymphocytes.     

High responsiveness of HLA-B57-restricted Gag-specific CD8+ T cells in vitro may contribute to the protective effect of HLA-B57 in HIV-infection

HLA-B57 has been shown to be associated with long-term asymptomatic HIV-1 infection. To investigate the biological mechanism by which the HLA-B57 allele could protect from HIV-1 disease, we studied both the number of CD8(+) T cells as well as CD8(+) T cell responsiveness directed to different HIV-1 Gag peptides presented by HLA-A2, -B8 or -B57. T cells specific for the HLA-B57 peptide KAFSPEVIPMF responded more readily and to a higher extend to antigenic stimulation in vitro than T cells specific for the HLA-A2 peptide SLYNTVATL or the HLA-B8 peptide EIYKRWII. This phenomenon was reproducible with T cells from individuals expressing HLA-B57 in combination with one or both of the other alleles and was persistent during long-term follow-up. Lower reactivity of A2- and B8-restricted T cells was not explained by mutations in the B8- or A2-restricted Gag-peptides. Moreover, no correlation between peptide mutation frequency and IFN-gamma production by the corresponding Gag-specific T cells was observed. In conclusion, functional differences were observed between T cells specific for HIV epitopes derived from the same protein presented by different HLA molecules. B57-restricted KAFSPEVIPMF-specific CD8(+) T cells have relatively high responsiveness, which could contribute to the protective effect of HLA-B57 in HIV infection.     

Clones of Human Cytotoxic T Lymphocytes Derived from an Allosensitized Individual: HLA Specificity and Cell Surface Markers

By planned immunization of a volunteer, two stable (greater than or equal to 6 months), specific, alloreactive cytolytic T-cell clones have been established from his peripheral blood lymphocytes. One clone reacts with all serologically defined HLA-Cw3 cells from our panel, whereas the other defines a split within the serological HLA-B40 specificity. The two cytotoxic clones are SmIg-negative, E-rosette positive, EA and EAC rosette-negative, HLA-A, -B and -C- positive, and also HLA-DR- or 'Ia like'-positive. In addition, they present very similar patterns of iodinated cell surface molecules as analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), contrasting with that of an EBV cell line derived from the same donor.